Methods for promoting production of viable seeds from apomictic guayule plants

ABSTRACT

Described herein are methods for producing guayule seeds, guayule plants, and products generated therefrom. More specifically, the disclosure provides methods for the production of viable seeds from apomictic guayule plants, seeds produced by such methods, plants grown from such seeds, plant parts, biomass, and biomaterials derived therefrom.

RELATED APPLICATIONS

This application is a Divisional of co-pending U.S. patent applicationSer. No. 16/721,385, which is a continuation of and claims the benefitand priority to U.S. patent application Ser. No. 15/571,001, fled onOct. 31, 2017, which is a U.S. National Phase Application of PCTInternational Application Number PCT/US2016029850, filed on Apr. 28,2016, designating the United States of America and published in theEnglish language, which is an International Application of and claimsthe benefit of priority to U.S. Provisional Application No. 62/156,148,filed on May 1, 2015. The disclosures of the above-referencedapplications are hereby expressly incorporated by reference in theirentireties.

FIELD

The present disclosure generally relates to the field of agriculture, inparticular to guayule seeds and methods for producing them. Inparticular, the disclosure provides methods for the production of viableseeds from guayule plants, including apomictic guayule plants. Seedsproduced by such methods, plants grown from such seeds, plant parts,biomass, and biomaterials derived therefrom are also provided.

BACKGROUND

Guayule (Parthenium argentatum A. Gray), a member of the familyCompositae (Asteraceae), is a desert shrub indigenous to thesouthwestern United States and northern Mexico. Guayule has beenlong-recognized as a particularly promising alternative source ofnatural rubber because of its potential for industrial-scale cultivationin the arid and semi-arid environments, and because the rubber harvestedfrom it shows unusual hypoallergenic properties. It is generallybelieved that the economics of guayule production will improvesignificantly if higher-yielding lines can be developed using reliableand rapid methods of selecting plants with the best possible traits thatwill be passed faithfully to their progeny. (Ray et al., IndustrialCrops and Products, 22:15-25, 2005).

Guayule has been readily manipulated by plant breeders for improvementbecause there exists many strains of wide genetic diversity withchromosome numbers of individual plants ranging from 2n=36 to 100 ormore. However, perhaps the biggest challenge in developing commercialgrade guayule hybrids through current plant breeding programs is thehighly complex reproduction biology of this crop plant which isassociated with its facultative nature of apomixis (e.g. asexualreproduction and sexuality coexisting). In fact, due to the facultativenature of apomixis in guayule, wild stands of this crop typicallycontain a natural polyploid series of diploids, triploids, andtetraploids; and under cultivation, individual plants have beenidentified with chromosome numbers up to octaploid (2N=8×=44). Whilediploids can reproduce predominantly sexually, they have had onlylimited use in current guayule breeding programs because they arelargely self-incompatible. Triploids and pentaploids are known to alsooccur in guayule but, from a breeding point of view, these individualsare considered dead ends because they are reproduce predominantlythrough apomixis and produce very little viable pollen. Therefore,although some triploids and pentaploids have been documented as havingrelatively high latex content, these were generally not pursued forcommercial latex production because a production field consisting ofonly apomictic triploids or pentaploids, albeit productive in terms oflatex, may have been unproductive in terms of seeds for subsequentplantation expansion.

As a result, most existing guayule germplasm consists primarily offacultatively apomictic reproducing tetraploid accessions, which havereceived most of the attention in breeding programs. However, theprocess of inbreeding of tetraploid parent lines often time can be veryconsuming, especially because apomixis and sexuality coexist in theseindividuals. In fact, due to the facultative nature of apomixis intetraploids, four classes of progeny generally exist. The origin andrelative chromosome numbers of these four classes from tetraploidparents, as will be discussed in further detail herein, illustrates thecomplexity of reproduction and the potential for release of geneticvariability in this species. In addition, the high amount ofheterozygosity in individual plants and the heterogeneous make-up ofguayule populations often results in the release of considerablevariation whenever sexual reproduction occurs. Consequently, most of theguayule varieties currently used in large-scale production lack geneticuniformity.

Thus, there is a long-standing and continuing need for new methods foroptimizing guayule breeding strategies for producing uniform hybridprogeny with agronomically desirable genotypes. This and other needs arediscussed by the present disclosure.

SUMMARY

This section provides a general summary of the disclosure, and is notcomprehensive of its full scope or all of its features.

Some alternatives disclosed herein relate to methods for promoting theproduction of viable hybrid guayule seeds from an interploidy cross. Themethods include selecting a female guayule plant and a pollinator plant,wherein the female guayule plant is apomictic and the pollinator plantis capable of producing fertile pollen; pollinating the female guayuleplant with pollen from the pollinator plant to produce seeds on theapomictic female guayule plant, where at least 5%, 10%, 15%, 20%, 25%,30%, 35%, 40%, 45%, 55%, 60%, 70%, 80%, 90%, 95%, or 100% of the hybridseeds are viable or where the percentage of viable hybrid seeds producedare within a range defined by any two of the aforementioned percentages.In some alternatives, the percentage of viable hybrid seeds is scored at35 days after germination.

In some alternatives, the pollinator plant is a tetraploid guayuleplant. In some alternatives, the female guayule plant is selected from atriploid guayule plant, a pentaploid guayule plant, and a heptaploidguayule plant. In some alternatives, the female guayule plant and saidpollinator plant belong to the same Parthenium species. In somealternatives, the Parthenium species is Parthenium argentatum. In somealternatives, the female guayule plant and the pollinator plant belongto two different Parthenium species. In certain alternatives, the femaleguayule plant is a Parthenium argentatum plant. In some alternative, thepollinator plant is of a species selected from the group consisting ofP. alpinum, P. argentatum, P. cineraceum, P. confertum, P. fruticosum,P. hysterophorus, P. incanum, P. integrifolium, P. ligulatum, P.rollinsianum, P. schottii, and P. tomentosum. In some alternatives, thepollinator plant is a Parthenium argentatum plant or a Partheniumincanum plant. In some alternatives, the pollinator plant is of aParthenium species that is different than Parthenium argentatum suchthat any F1 hybrid off types can be conveniently identified. In somealternatives, the pollinator plant is of a Parthenium species that isdifferent than Parthenium argentatum such that F1 hybrid off-types canbe conveniently identified based on inheritance of leaf morphologicaldifferences from the respective pollinator plants. In some alternatives,at least one of the female guayule plant or the pollinator plant ispre-selected for enhanced plant productivity. In some alternatives, atleast one of the female guayule plant or the pollinator plant isclonally propagated prior to pollination step.

Some alternatives disclosed herein relate to methods for producinghybrid guayule seeds from an interploidy cross between female guayuleplants from at least one female guayule line and pollinator plants fromat least one pollinator plant line, in which the hybrid seeds having apercentage of viability above a predetermined percentage of viability.The methods include selecting the at least one female guayule line andthe at least one pollinator plant line; determining a production ratioof female plants to pollinator plants to produce hybrid seeds having apercentage of viability above said predetermined percentage ofviability; planting the at least one female guayule line and the atleast one pollinator plant line in pollinating proximity in theproduction ratio in a production field; and allowing pollination of atleast one of the female guayule plants with pollen from at least one ofthe pollinator plants, whereby the hybrid guayule seeds are produced. Incertain alternative, the methods further include harvesting the hybridguayule seeds.

In some alternatives, the female guayule plants include plants from asingle female line. In some alternatives, the female guayule plantsinclude plants from a plurality of female lines. In some alternatives,the at least one of said guayule female lines is a cloned apomicticguayule line. In some alternatives, the pollinator plants include plantsfrom a single pollinator line. In some alternatives, the pollinatorplants include plants from a plurality of pollinator lines. In somealternatives, the predetermined percentage of viability is 5%, 10%, 15%,20%, 25%, 30%, 35%, 40%, 45%, 55%, 60%, 70%, 80%, 90, 95%, or 100%, orwherein the predetermined percentage of viability is within a rangedefined by any two of the aforementioned percentages. In somealternatives, the percentage of viable hybrid seeds is scored at 35 daysafter germination. In some alternatives, the production ratio of femaleplants to pollinator plants ranges from 10:1 to 1:5. In somealternatives, the production ratio of female plants to pollinator plantsis 5:1, 4:1, 3:1, 2:1, or 1:1 or within a range defined by any two ofthe aforementioned ratios. In some alternatives, the selecting of atleast one pollinator plant line includes selecting a pollinator linehaving high self-incompatibility or high production of viable pollen. Insome alternatives, the planting includes planting the female guayuleplants and the pollinator plants in multiple rows, in which each row ofpollinator plants is interspersed with a plurality of rows of femaleguayule plants. In some alternatives, the plurality of rows of femaleguayule plants is 2 to 5 rows.

Some alternatives of the disclosure relate to viable hybrid guayuleseeds produced by a method disclosed herein. In some alternatives, theviable plant seeds are produced by selecting a female guayule plant anda pollinator plant, wherein the female guayule plant is apomictic andthe pollinator plant is capable of producing fertile pollen; pollinatingthe female guayule plant with pollen from the pollinator plan to produceseeds on the apomictic female guayule plant, where at least 5%, 10%,15%, 20%, 25%, 30%, 35%, 40%, 45%, 55%, 60%, 70%, 80%, 90%, 95%, or 100%of the hybrid seeds are viable or where the percentage of viable hybridseeds produced are within a range defined by any two of theaforementioned percentages. In some alternatives, the viable plant seedsare produced from an interploidy cross between female guayule plantsfrom at least one female guayule line and pollinator plants from atleast one pollinator plant line, in which the hybrid seeds having apercentage of viability above a predetermined percentage of viability.In some alternatives, the viable plant seeds are produced by (i)selecting at least one female guayule line and at least one pollinatorplant line; (ii) determining a production ratio of female plants topollinator plants to produce hybrid seeds having a percentage ofviability above said predetermined percentage of viability; (iii)planting the at least one female guayule line and the at least onepollinator plant line in pollinating proximity in the production ratioin a production field; and (iv) allowing pollination of at least one ofthe female guayule plants with pollen from at least one of thepollinator plants, whereby the hybrid guayule seeds are produced.

Some alternatives disclosed herein relate to plants grown from thehybrid guayule seeds described herein. In some alternatives, the plantsfurther include a transgene. In some alternatives, the transgene confersa trait selected from the group consisting of: abiotic stress tolerance,biotic stress tolerance, disease resistance, high latex yield, highoverall rubber yield, high productivity, high resin yield, improvednitrogen use efficiency, improved water use efficiency, and plant vigor,or any combination of the aforementioned traits. Some alternativesrelate to seeds, reproductive tissues, vegetative tissues, plant parts,biomass, or progeny of a plant disclosed herein. In some alternatives,the part of the plant is selected from the group consisting of ananther, an axillary bud, a cell, an embryo, a female gametophyte, afilament, a flower, a inflorescence, a leaf, a male gametophyte, ameristem, an ovary, an ovule, a petal, a pistil, a pollen grain, aprotoplast, a root, a seed, a sepal, a stamen, a stem, a stigma, astyle, a terminal bud, a cell of said plant in culture, a tissue of saidplant in culture, an organ, a cutting, an explant, and a callus.

Some alternatives disclosed herein relate to methods for producing aproduct, including obtaining a plant or parts thereof as described inthe present disclosure. Also disclosed, in some alternatives, are plantderived-products that are produced by the methods disclosed herein. Insome alternatives, the plant-derived product is selected from the groupconsisting of latex, resin, fatty acid triglycerides, terpenes,sesquiterpenes, and waxes.

Some alternatives disclosed herein relate to a latex product derivedfrom the latex disclosed herein, wherein the latex product is selectedfrom the group consisting of medical gloves, surgical gloves, elasticbands, elastic traps, condom, automobile tires, truck tires, airplanetires, and wet suits.

The foregoing summary is illustrative only and is not intended to be inany way limiting. In addition to the illustrative aspects, alternatives,and features described above, further aspects, alternatives, andfeatures will become apparent by reference to the drawings and thefollowing detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 : Events recorded by the flow cytometer plotted by forwardscatter (y-axis) and DNA fluorescence (x-axis). A nuclei gate was usedto remove background debris from further analysis.

FIG. 2 : Histogram of fluorescence values from gated events in FIG. 1 .The gate provided the median fluorescence value for nuclei in thesample.

FIG. 3 : Fluorescent values of nuclei samples acquired from the F1seedlings of each caged cross. Values were ordered from left to right byincreasing fluorescence regardless of replicate or seedling ID.Horizontal dotted lines represented median fluorescent values ofintraspecific controls (diploid, triploid, tetraploid, pentaploid).

FIG. 4 : Events recorded by the flow cytometer plotted by forwardscatter (y-axis) and DNA fluorescence (x-axis). A nuclei gate is used toremove background debris from further analysis.

FIG. 5 : Histogram of fluorescence values from gated events in FIG. 4 .The gate gives the median fluorescence value for nuclei in the sample.

FIG. 6 : Fluorescence of intraspecific guayule controls (2×, 3×, 4× fromleft to right). Dotted lines correspond to median fluorescent values foreach ploidy level.

FIG. 7 : Fluorescence of F1 seedlings resulting from the pollination ofa pentaploid guayule by a mariola plant. Dotted lines correspond tomedian fluorescent values for each ploidy level.

DETAILED DESCRIPTION OF THE DISCLOSURE

In the following detailed description, reference is made to theaccompanying drawings, which form a part hereof. In the drawings,similar symbols typically identify similar components, unless contextdictates otherwise. The illustrative alternatives described in thedetailed description, drawings, and claims are not meant to be limiting.Other alternatives may be used, and other changes may be made, withoutdeparting from the spirit or scope of the subject matter presented here.It will be readily understood that the aspects of the presentdisclosure, as generally described herein, and illustrated in theFigures, can be arranged, substituted, combined, and designed in a widevariety of different configurations, all of which are explicitlycontemplated and make part of this disclosure.

Unless otherwise defined, all terms of art, notations and otherscientific terms or terminology used herein are intended to have themeanings commonly understood by those of skill in the art to which thisdisclosure pertains. In some cases, terms with commonly understoodmeanings are defined herein for clarity and/or for ready reference, andthe inclusion of such definitions herein should not necessarily beconstrued to represent a substantial difference over what is generallyunderstood in the art. Many of the techniques and procedures describedor referenced herein are well understood and commonly employed usingconventional methodology by those skilled in the art.

SOME DEFINITIONS

The singular form “a”, “an”, and “the” include plural references unlessthe context clearly dictates otherwise. For example, the term “amolecule” includes one or more molecules, including mixtures thereof. “Aand/or B” is used herein to include all of the following alternatives:“A”, “B”, and “A and B”.

The term “about”, as used herein, has its ordinary meaning ofapproximately. If the degree of approximation is not otherwise clearfrom the context, “about” means either within plus or minus 10% of theprovided value, or rounded to the nearest significant figure, in allcases inclusive of the provided value. Where ranges are provided, theyare inclusive of the boundary values.

“Apomixis”, as used herein, in flowering plants is defined as theasexual formation of a seed from the maternal tissues of the ovule,avoiding the processes of meiosis and fertilization, leading to embryodevelopment. All known mechanisms of apomixis share three developmentalcomponents: the generation of a cell capable of forming an embryowithout prior meiosis (apomeiosis); the spontaneous,fertilization-independent development of the embryo (parthenogenesis);and the capacity to either produce endosperm autonomously or to use anendosperm derived from fertilization. As used herein, “obligate apomict”and grammatical equivalents thereof refer to a solitary plant thatreproduces only by apomixis. The term “facultative apomict” andgrammatical equivalents thereof refer to a solitary plant thatreproduces both sexually and apomictically.

“Biomass”: As used herein, plant biomass refers to the amount of (e.g.,measured in grams of air-dry tissue) of a harvestable plant tissueproduced from the plant in a growing season, which could also determineor affect the plant yield or the yield per growing area. Non-limitingexamples of such harvestable plant tissues include leaves, stems, andreproductive structures, or all plant tissues such as leaves, stems,roots, and reproductive structures.

As used herein, the term “cultivar” or a “variety” refers to a group ofsimilar plants that belong to the same species and that, by structuralfeatures and performance, may be distinguished from other varietieswithin the same species. Two essential characteristics of a variety areidentity and reproducibility. Identity is necessary so that the varietymay be recognized and distinguished from other varieties within the cropspecies. The distinguishing features may be morphologicalcharacteristics, molecular markers, color markings, physiologicalfunctions, disease reaction, or performance. Most agricultural varietiesare pure for the characteristic or for those characteristics thatidentify the variety; per se. Reproducibility is needed in order thatthe characteristic(s) by which the variety is identified will bereproduced in the progeny. For the purpose of this disclosure,therefore, the terms “cultivar” and “variety” are used interchangeablyto refer to a group of plants within a species (for example, Partheniumargentatum) that share certain constant characters which separate themfrom the typical form and from other possible varieties within thatspecies. While possessing at least the distinctive trait, a “variety” ofthe disclosure also may be characterized by a substantial amount ofoverall variation between individuals within the variety, basedprimarily on the Mendelian segregation of traits among the progeny ofsucceeding generations. On the other hand, “cultivar” or “variety” alsocan denote a cloned line, because a Parthenium argentatum cultivar mayindividually be reproduced asexually, via stem cuttings, and all of theclones would be essentially identical genetically.

As used herein, a “line” refers to a population of plants derived from asingle cross, backcross or selfing. The individual offspring plants arenot necessarily Identical to one another. As distinguished from a“variety,” a “line” displays less variation between individuals,generally (although not exclusively) by virtue of several generations ofself-pollination. For purposes of this disclosure, a “line” is definedsufficiently broadly to include a group of plants vegetatively orclonally propagated from a single parent plant, using stem cuttings ortissue culture techniques. “Vegetative propagation”, as used herein,refers to asexual propagation of the plant that is accomplished bytaking and propagating cuttings, by grafting or budding, by layering, bydivision of plants, or by separation of specialized structure, such asstem, roots, tubers, rhizomes, or bulbs.

The term “breeding line”, as used herein, refers to a line of acultivated crop having commercially valuable or agronomically desirablecharacteristics, as opposed to wild varieties or landraces. The termincludes reference to an elite breeding line or elite line, whichrepresents a line of plants used to produce commercial F1 hybrids. Anelite breeding line is obtained by breeding and selection for superioragronomic performance comprising a multitude of agronomically desirabletraits.

The term “hybrid”, as used herein, refers to any offspring of a crossbetween two genetically non-identical individuals. The parental plantsmay be related, as in production of a modified single cross, orunrelated. F1 hybrid, as used herein, refers to the first generationprogeny of the cross of two genetically dissimilar plants.

As used herein, the expressions “latex content”, “rubber content”, and“resin content” refer to the amount of latex, rubber, and resinrespectively, in a given plant organ or tissue, such as the stem and istypically expressed as percentage of dry weight (for example at 10%humidity of biomass) or wet weight. It should be noted that latex,rubber. and resin content is affected by intrinsic latex, rubber, andresin production of a tissue (e.g., stem, leaf), as well as the mass orsize of the latex-producing tissue per plant or per growth period. Insome alternatives, increase in latex, rubber, or resin content of theplant can be achieved by increasing the size/mass of a plant's tissue(s)which contains latex, rubber, and resin per growth period. Thus,increased latex, rubber, and/or resin content of a plant can be achievedby increasing the yield, growth rate, biomass and vigor of the plant.

The term “plant part” refers to any part of a plant including, but notlimited to, organelles, single cells and cell tissues such as plantcells that are intact in plants, cell clumps and tissue cultures fromwhich guayule plants can be regenerated. Examples of plant partsinclude, but are not limited to, single cells and tissues from pollen,ovules, leaves, embryos, roots, root tips, tubers, anthers, flowers,fruits, stems shoots, and seeds; as well as pollen, ovules, leaves,embryos, roots, root tips, anthers, flowers, fruits, stems, shoots,scions, rootstocks, seeds, tubers, protoplasts, and calli. The two mainparts of plants grown in some sort of media, such as soil, are oftenreferred to as the “above-ground” part, also often referred to as the“shoots”, and the “below-ground” part, also often referred to as the“roots”.

As used herein, “progeny” include descendants of a particular plant orplant line. Progeny of an instant plant include seeds formed on F1, F2,F3, F4, F5, F6 and subsequent generation plants, or seeds formed on BC1,BC2, BC3, and subsequent generation plants, or seeds formed on F1BC1,FiBC2, FiBC3, and subsequent generation. plants. The designation F1refers to the progeny of a cross between two parents that aregenetically distinct. The designations F2, F3, F4, F5 and F6 refer tosubsequent generations of self- or sib-pollinated progeny of an F1plant.

As will be understood by one having ordinary skill in the art, for anyand all purposes, such as in terms of providing a written description,all ranges disclosed herein also encompass any and all possiblesub-ranges and combinations of sub-ranges thereof. Any listed range canbe easily recognized as sufficiently describing and enabling the samerange being broken down into at least equal halves, thirds, quarters,fifths, tenths, etc. As a non-limiting example, each range discussedherein can be readily broken down into a lower third, middle third andupper third, etc. As will also be understood by one skilled in the artall language such as “up to,” “at least,” “greater than,” “less than,”and the like include the number recited and refer to ranges which can besubsequently broken down into sub-ranges as discussed above. Finally, aswill be understood by one skilled in the art, a range includes eachindividual member. Thus, for example, a group having 1-3 articles refersto groups having 1, 2, or 3 articles. Similarly, a group having 1-5articles refers to groups having 1, 2, 3, 4, or 5 articles, and soforth.

The present disclosure generally describes methods for generating viablehybrid seeds from apomictic guayule plants. Seeds produced by suchmethods, plants grown from such seeds, plant parts, biomass, andbiomaterials derived therefrom are also provided.

APOMIXIS

In angiosperms, two pathways of reproduction through seed exist: sexualor amphimictic and asexual or apomictic. The latter is largely exploitedby seed companies for crop production and breeding new varieties,whereas the latter is receiving continuously increasing attention fromboth scientific and industrial sectors. The term apomixis is generallyinterpreted as the replacement of sexual reproduction by various formsof asexual reproduction. Mechanistically, apomixis is a geneticallycontrolled reproductive method in plants where the embryo is formedwithout union of an egg and a sperm. (Bicknell and Koltunow, Plant Cell,Vol. 16, S228-245, 2004). Apomixis affects both megasporegenesis andmegagametogenesis, but typically does not alter pollen formation.Meiosis still occurs normally in the anthers, and viable, reduced pollenis usually produced in both aposporous and diplosporous apomicts. Thereare three basic types of apomictic reproduction: 1) apospory-embryodevelops from a chromosomally unreduced egg in an embryo sac derivedfrom the nucellus, 2) diplospory-embryo develops from an unreduced eggin an embryo sac derived from the megaspore mother cell, and 3)adventitious embryony-embryo develops directly from a somatic cell. Inmost forms of apomixis, pseudogamy or fertilization of the polar nucleito produce endosperm is necessary for seed viability. Apomixis isconsidered to be facultative when some progeny also result from either anormal meiosis and/or a normal fertilization of the egg cell, and as aresult, a facultative apomictic plant can produce various frequencies ofmaternal progenies. Apomixis is considered to be obligate when theprogeny is 100% maternal.

Apomixis has an important potential impact on agricultural and foodproduction by reducing cost and breeding time, and avoiding thecomplications that are typical of sexual reproduction (for example,incompatibility barriers) and vegetative propagation (for example, viraltransfer). In addition, in outcrossing species that reproduce sexually,alleles disseminate in the offspring; thus, the optimal genotype is losttogether with the desired trait. In contrast, apomixis is a reproductiveprocess that bypasses female meiosis and syngamy to produce embryosgenetically identical to the maternal parent, and therefore apomixis cancause any genotype, regardless of how heterozygous, to breed true, e.g.,progeny of highly adaptive or hybrid genotypes with apomicticreproduction would maintain their genetic fidelity throughout repeatedlife cycles. Therefore, the genotype of every apomictic would be fixedin the F1 generation and every apomictic genotype from a cross has thepotential of being a cultivar. Gene combinations and vigor would not belost as in each segregating generation of sexual F1 hybrids. Themaintenance of elite genotypes would be therefore easier and moreefficient. In addition to fixing hybrid vigor, apomixis can makepossible commercial hybrid production in crops where efficient malesterility or fertility restoration systems for producing hybrids are notknown or developed. Further, apomixis could have a major impact incommercial hybrid production systems by simplifying hybrid seedproduction and therefore making hybrid development more efficient.

GUAYULE

Guayule, a member of the family Compositae (Asteraceae), is axerophytic, woody perennial shrub indigenous to the southwestern UnitedStates and northern Mexico. In addition to producing hydrocarbon rubberpolymers during the winter (Cornish and Backhaus, Industrial Crops andProducts, 17:83-92, 2003), guayule produces and stores a high-energyhydrocarbon terpenoid resin in specialized resin vessels throughout theyear (Coffelt et al., Industrial Crops and Products, 29:255-260, 2009).Guayule (Parthenium argentatum Gray) is so named because of a silverysheen on its gray-green leaves which are covered in a drought protectingwhite wax. Guayule flowers are pollinated by wind and by insects.Guayule plant is generally a bushy perennial shrub with alternate narrowleaves along the stem. It bears a canopy inconspicuous, hardy andbetween two and three feet high. The plant generally can survive 30-40years under desert conditions. Descriptions of guayule cultivationpractices have been reviewed extensively by Thompson and Ray (PlantBreed Rev. 6:93-165, 1989) and Ray D T (Guayule: A source of naturalrubber. p. 338-343. In: J. Janick and J. E. Simon (eds.), 1993, NewCrops. Wiley, New York). Guayule has been long-recognized as aparticularly promising alternative source of natural rubber andbioactive terpenoid compounds, because of its potential forIndustrial-scale cultivation in the arid and semi-arid environments, andbecause the rubber harvested from it shows unusual hypoallergenicproperties. It is generally believed that the economics of guayuleproduction will improve significantly if higher-yielding lines can bedeveloped using reliable and rapid methods of selecting plants with thebest possible traits that will be passed faithfully to their progeny.Ray et al., J. Amer. Soc. Hort. Sci. 132(2) 213-218, 2007; (Ray et al.,Industrial Crops and Products, 22:15-25, 2005).

However, perhaps the biggest challenge in developing commercial gradeguayule hybrids through current plant breeding programs is the highlycomplex reproduction biology of this crop plant, which is associatedwith its facultative nature (e.g. apomixis and sexuality coexisting) andthe high amount of heterozygosity in individual plants and theheterogeneous make-up of populations, resulting in the release ofconsiderable variation whenever sexual reproduction (amphimixis) occurs.Most of the guayule varieties currently used in large-scale productionare heterozygous at many loci and thus lack genetic uniformity. As such,a sexual cross between two genetically dissimilar parents typicallyresults in a heterogeneous collection of F1 hybrids that not onlysegregate genotypically, but also show dramatic phenotypic differences.Further, the process of inbreeding of parent lines often time can bevery consuming, especially because apomixis and sexuality coexist. Infact, diploid guayule plants reproduce predominantly sexually, andpolyploids reproduce by facultative apomixis. Due to the facultativenature of guayule, wild stands of guayule typically contain a naturalpolyploid series of diploids (2N=2×=36), triploids (2N=3×=54) andtetraploids (2N=4×=72), and under cultivation, individual plants havebeen identified with chromosome numbers up to octaploid (2N=8×=144).Another reproductive feature is reported to occur frequently inpolyploid guayule is haploidy, which is the reduction of chromosomenumber from, for example, 2N to 1N. In this instant, the egg cell bas areduced chromosome number because meiosis has occurred, but the stimulusfor apomictic development still exists and the egg in the reducedcondition produces a new haploid plant. In addition, to furthercomplicate current breeding efforts, guayule also has a sporophyticsystem of self-incompatibility and many plants contain B- orsupernumerary chromosomes (Ray et al. 2007, supra; Thompson and Ray,Breeding guayule, Plant Breed Rev., 6:93-165, 1989).

Most existing guayule germplasm consists primarily of apomicticallyreproducing tetraploid accessions, which have received most of theattention in breeding programs. The tetraploid plants are generally muchlarger and more vigorous than the diploids, and they readily produceseeds through apomixis. Fields of primarily tetraploid plants could beused to not only produce latex/rubber but could also be used for seedproduction. Breeders have performed mass selections on tetraploid linesto identify individuals with higher latex/rubber yields. These can bepropagated by cuttings or by the facultatively apomictic-derived seeds.However, the process of inbreeding of parent lines often time can bevery consuming, especially because apomixis and sexuality coexist. Infact, the facultative nature of apomixis in tetraploid plants results infour classes of progeny. The origin and relative chromosome numbers ofthese four classes from tetraploid parents illustrates the complexity ofreproduction and the potential for release of genetic variability inthis species. The predominant class of progeny arises from non-reductionof the megaspore mother cell (MMC), without fertilization. These areapomictic tetraploid progeny and are identical generically to thematernal parent. Progeny from fertilized, unreduced MMCs include plantswith increased ploidy levels. In this example, these progeny would behexaploid (2N=6×=108). Polyhaploid (2N=2×=36) plants are the result ofmeiotic reduction of the tetraploid MMC, and embryo development withoutfertilization. The final class would be amphimictic tetraploid(2N=4×=72) progeny that arises from normal reduction and fertilization.Thus, there are two reproductive modes that produce tetraploid progeny,one by apomixis and the other by sexual reproduction. As a result, aplant breeder cannot easily differentiate between the apomictic andsexual progeny based on chromosome number alone. The remaining twoprogeny classes vary in chromosome number from the parental populationand therefore can be identified relatively easy.

On the other hand, triploids and pentaploids are known to occur inguayule, but have had only limited use in current guayule breedingprograms. Although some triploids and pentaploids have been documentedas having a relatively high latex content, these were generally notpursued for commercial latex production because a field of onlytriploids or pentaploids, although productive in terms of latex, mayhave been unproductive in terms of seeds for subsequent plantationexpansion. This is because, similar to other polyploids, triploid (3×)and pentaploid (5×) guayule reproduce apomictically. From a breedingpoint of view, these individuals are considered dead ends because theyreproduce predominantly through apomixis and produce very little viablepollen. In the case of triploids, a small percentage of off-types, e.g.,pentaploid (5×) seeds can also occur at low frequencies. Triploid andpentaploid guayule plants generally can produce flowers and set seed.However in the absence of fertile pollen from tetraploid (4×)pollinator, the endosperm development is defective and while many of theseeds apomictically produced by triploid and pentaploid guayule plantswith unfertilized endosperm can germinate, none of the seedlings survivepast day 30 after germination. From these studies, a method forgenerating viable seed was devised and disclosed herein.

As described in greater detail in the Examples below, Applicants havedeveloped a breeding strategy that takes advantage of all these subtlereproductive nuances in guayule, and combines them into methods thatpermit the production of viable hybrid seeds from triploid andpentaploid guayule plants.

In particular, according to some alternatives disclosed herein,Applicants have demonstrated that the production of viable apomicticseed production in triploids and pentaploids require the presence offertile pollen. While Applicants do not wish to be bound by any theory,it is believed that the presence of pollen from fertile plants,particularly tetraploid plants, help assure the completion of seeddevelopment in the apomictically produced seeds on triploid andpentaploid plants. While the embryo is not fertilized, pollen fromfertile plants assures the fertilization of the polar nuclei andsubsequent completion of the endosperm development in theseapomictically produced seeds.

SEED PRODUCTION METHODS

In one aspect, some alternatives of the present disclosure relate tomethods for promoting the production of viable hybrid guayule seeds froman interploidy cross. In some alternatives, the methods include a stepof selecting a female guayule plant and a pollinator plant, where thefemale guayule plant is apomictic and the pollinator plant is capable ofproducing fertile pollen. Generally, the female plant can be any guayuleplant capable of producing an embryo and can be, for example, a diploidguayule plant, a triploid guayule plant, a tetraploid guayule plant, apentaploid guayule plant, a hexaploid guayule plant, a heptaploidguayule plant, or an octaploid guayule plant. In some preferredalternatives, the female plant can be selected from the group consistingof a triploid guayule plant, a pentaploid guayule plant, or andheptaploid guayule plant. In some particularly preferred alternatives,the female plant can be a triploid guayule plant. In some particularlypreferred alternatives, the female plant can be a pentaploid guayuleplant. In some alternative of the methods disclosed herein, the malepollinator plant can generally be any plant capable of producing fertilepollen. Non-limiting examples of suitable pollinator plants include ahaploid guayule plant, a diploid guayule plant, a triploid guayuleplant, a tetraploid guayule plant, a pentaploid guayule plant, ahexaploid guayule plant, a heptaploid guayule plant, or an octaploidguayule plant. In some preferred alternatives, the male pollinator canbe a tetraploid guayule plant.

In some alternatives of the methods disclosed herein, a second stepcomprises pollinating the female guayule plant with pollen from thepollinator plant to produce seeds on the apomictic female guayule plant.In addition, this step can optionally comprise preventingself-pollination of the plants, e.g., preventing the ovules of a plantfrom being fertilized by pollen of the same plant or of any plant of thesame plant cultivar or variety. This can be done, for example, byemasculating the flowers of the female plant, (e.g., treating ormanipulating the flowers so as to prevent pollen production, in order toproduce an emasculated parent female guayule plant).Self-incompatibility systems may also be used in some hybrid productionschemes for the same purpose. Typically, self-incompatible plants stillshed viable pollen and can pollinate plants of other varieties but areincapable of pollinating themselves or other plants of the same variety.

In some embodiments, the female guayule plant is pollinated with pollenfrom the pollinator plant to produce seeds on the apomictic femaleguayule plant in a manner that at least about 5%, at least about 10%, atleast about 15%, at least about 20%, at least about 25%, at least about30%, at least about 35%, at least about 40%, at least about 45%, atleast about 55% of the hybrid seeds are viable. In some embodiments, thefemale guayule plant is pollinated with pollen from the pollinator plantto produce seeds on the apomictic female guayule plant in a manner thatat least about 60%, at least about 70%, at least about 80%, at leastabout 90%, at least about 95%, or at least about 100% of the hybridseeds are viable. In some embodiments, the female guayule plant ispollinated with pollen from the pollinator plant to produce seeds on theapomictic female guayule plant in a manner that the percentage of viablehybrid seeds produced are within a range defined by any two of theaforementioned percentages. In some particular alternatives of themethods disclosed herein, about 13% of the hybrid seeds are viable. Insome alternatives, about 35% of the hybrid seeds are viable. In somealternatives, about 40% of the hybrid seeds are viable. In somealternatives, about 65% of the hybrid seeds are viable.

The percentage of viable hybrid seeds can be scored at differentdevelopmental stages and can be, for example, scored at 15, 20, 30, 40,50 days after germinations. In some alternatives, the percentage ofviable hybrid seeds is scored at 35 days after germination.

In principle, the methods disclosed herein can be deployed for theproduction of viable hybrid seeds of any Parthenium species. SuitableParthenium species to be used include, but not limited to P. alpinum, P.argentatum, P. cineraceum, P. confertum, P. fruticosum, P.hysterophorus, P. incanum, P. integrifolium, P. ligulatum, P.rollinsianum, P. schottii, and P. tomentosum. Preferred Partheniumspecies include Parthenium argentatum or a Parthenium incanum.Particularly preferred Parthenium species to be used in the methodsdisclosed herein is Parthenium argentatum. Non-limiting examples ofpreferred P. argentatum varieties and cultivars include AZ-2, AZ-4,AZ-R2, AZ-11591, ARIZ-101, GH-9, UCR-7, UCR-1, USDA-10, USDA-5, USDA-7,ALI-10, USS2X, G-89, G-97, C-244, USDA Cal-6, USDA Cal-7, USDA-593,USDA-N565, and USDA-N396. Other non-limiting examples of preferred P.argentatum varieties and cultivars include YX-5-03-bLr-02 andYX-5-03-bLr-02 (Yulex Corp.). Other non-limiting examples of preferredP. argentatum varieties and cultivars include PA 11-2, PA 11-3, PA 11-4,PA 11-8, PA 10-4, PA 10-14, PA 10-16, PA 10-18, and PA 10-23 (Panaridus,LLC).

In some alternatives of the methods disclosed herein, the female guayuleplant and the pollinator plant selected for the interploidy cross belongto the same Parthenium species. In some alternatives, the female guayuleplant and the pollinator plant selected for the interploidy cross belongto different Parthenium species such that any F1 hybrid off types fromthe interploidy cross can be conveniently identified. In somealternatives, the female guayule plant is a Parthenium argentatum plant.In some alternatives, the pollinator plant is of a species selected fromthe group consisting of P. alpinum, P. argentatum, P. cineraceum, P.confertum, P. fruticosum, P. hysterophorus, P. incanum, P.integrifolium, P. ligulatum, P. rollinsianum, P. schottii, and P.tomentosum. In some preferred alternatives, the pollinator plant is aParthenium argentatum plant or a Parthenium incanum plant. In someparticularly preferred alternatives, the female plant is a Partheniumargentatum plant and the pollinator plant is of a Parthenium speciesthat is different than Parthenium argentatum such that any F1 hybrid offtypes from the interploidy cross can be conveniently identified.

In some alternatives of the methods disclosed herein, at least one ofthe parental female plant and the male pollen donor can be pre-selectedfor enhanced plant productivity. As used herein, “enhanced plantproductivity” refers to any aspect of a plant altered for a “desiredbenefit,” such as increasing an agriculturally desirable trait. “Desiredbenefit” also refers to any effect on a plant to confer a benefit tohumans. As used herein, “agriculturally desirable trait” refers to anyqualitative and quantitative agricultural trait, such as highproductivity, crop yield, biomass, resistance to pathogens, resistanceto pests, resistance to environmental changes, for example, drought,etc. In other words, a desirable trait is any characteristic worthobtaining. In some alternatives, the guayule plants selected asdescribed above are intercrossed with one another, followed by progenytesting to evaluate general combining abilities (intercrossability).

In some alternatives, at least one of the parental female plant and themale pollen donor can be clonally propagated prior to pollination step.The clonal propagation can generally be any known clonal propagationtechniques and can be, for example, tissue-culture, rooted cutting, stemcutting, stake cutting, or any of other means of vegetative propagation.

In one aspect, some alternatives of the present disclosure relate tomethods for producing hybrid guayule seeds from an interploidy crossbetween female guayule plants from at least one female guayule line andpollinator plants from at least one pollinator plant line, in which thehybrid seeds having a percentage of viability above a predeterminedpercentage of viability. In some alternatives, the methods includeselecting the at least one female guayule line and the at least onepollinator plant line; determining a production ratio of female plantsto pollinator plants to allow for production of hybrid seeds having apercentage of viability above said predetermined percentage ofviability; planting the at least one female guayule line and the atleast one pollinator plant line in pollinating proximity in theproduction ratio in a production field; and allowing pollination of atleast one of the female guayule plants with pollen from at least one ofthe pollinator plants, whereby the hybrid guayule seeds are produced.The term “pollinating proximity” as used herein refers to the distancethat two plants or two rows of plants can be planted from each other butcan still cross-pollinate.

In some alternatives, selection of female guayule line and thepollinator plant line takes into account both agronomic factors andfactors that affect percentage of Intercrossability (e.g., generalcombining abilities), hybrid seed yield, and hybrid seed viability. Theterm “intercrossable”, as used herein, refers to the ability to yieldprogeny plants after making crosses between parental plants. In someexemplary alternatives, these agronomic factors and factors include:female to pollinator ratio, seed yield from pollinator plants, seedyield from female plants, female to pollinator seed yield differential,female to pollinator seed size differential, high seed yield withincreasing female to pollinator plant ratio, low P.P.I. (PollenProduction Index), large seed size, pollination power in pollinatorplants, high production of viable pollen in pollinator plants, and highself-incompatibility in pollinator plants. In further aspects of thedisclosure, these factors include desirable agronomic traits such asdisease resistance, insect resistance, and abiotic stress resistance.

In certain alternative, the methods disclosed herein further includeharvesting the hybrid guayule seeds.

In some alternatives, the female guayule plants include plants from asingle female line. In some alternatives, the female guayule plantsinclude plants from a plurality of female lines. In principle, themethods disclosed herein can generally include plants from any number offemale lines and can include, for example, plants from at least 2, atleast 3, at least 4, at least 5, at least 6, at least 7, at least 8, atleast 9, at least 10 female lines. In some alternatives, the at leastone of said guayule female lines is a cloned apomictic guayule line,e.g. a line including guayule plants vegetatively or clonally propagatedfrom a single parent plant, using stem cuttings or tissue culturetechniques. In some alternatives, the pollinator plants include plantsfrom a single pollinator line. In some alternatives, the pollinatorplants include plants from a plurality of pollinator lines. Generally,the methods can include plants from any number of female lines and caninclude, for example, plants from at least 2, at least 3, at least 4, atleast 5, at least 6, at least 7, at least 8, at least 9, at least 10pollinator lines. In some alternatives, the predetermined percentage ofviability is 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 55%, 60%, 70%,80%, 90, 95%, or 100%, or wherein the predetermined percentage ofviability is within a range defined by any two of the aforementionedpercentages. In some alternatives, the percentage of viable hybrid seedsis scored at 35 days after germination. In some alternatives, theproduction ratio of female plants to pollinator plants ranges from 10:1to 1:5. In some alternatives, the production ratio of female plants topollinator plants is 5:1, 4:1, 3:1, 2:1, or 1:1 or within a rangedefined by any two of the aforementioned ratios. In some alternatives,the selecting of at least one pollinator plant line includes selecting apollinator line having high self-incompatibility or high production ofviable pollen. In some alternatives, the planting includes planting thefemale guayule plants and the pollinator plants in multiple rows, inwhich each row of pollinator plants is interspersed with a plurality ofrows of female guayule plants. The plurality of rows of female guayuleplants can generally, 16 rows of be any number of rows and can be, forexample, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 rows of femaleguayule plants, or within a range defined by any two of theaforementioned. For example, in some alternatives, the plurality of rowsof female guayule plants can be 2 to 10 rows, 5 to 15 rows, 3 to 9 rows,4 to 8 rows, 5 to 10 rows. In some alternatives, the plurality of rowsof female guayule plants can be 2 to 5 rows.

In one aspect, some alternatives of the present disclosure relate toviable hybrid guayule seeds that are produced by a method in accordancewith some alternatives disclosed herein.

In a related aspect, some alternatives of the present disclosure relateto a guayule plant that is grown from a seed produced by a method inaccordance with some alternatives disclosed herein. As use herein,phrases such as “grown from the seed” or “grown the seed” includetechniques and practices known in the art including, but are not limitedto, embryo rescue, isolation of cells from seed for use in tissueculture, as well as traditional growing methods. Descriptions of currentguayule cultivation practices have been reviewed extensively.Information in this regard can be found in, for example, Thompson andRay (Breeding guayule. Plant Breed Rev. 6:93-165, 1989, and in GuayuleNatural Rubber, edited by Whitworth and Whitehead (1991).

As used herein, the term “tissue culture” refers to a compositioncomprising isolated plant cells of the same or a different type or acollection of such cells organized into parts of a plant, in which thecells are propagated in a nutrient medium under controlled conditions.Non-limiting examples of tissue cultures include plant protoplasts,plant cell tissue culture, culture microspores, plant calli, plantclumps, and the like.

In one aspect, the present disclosure further provides a seed, areproductive tissue, a vegetative tissue, a plant part, a biomass, orprogeny of a guayule hybrid plant disclosed herein. In some alternativesof this aspect, provided herein is a plant part of a guayule hybridplant disclosed herein, wherein the plant part is selected from thegroup consisting of an anther, an axillary bud, a cell, an embryo, afemale gametophyte, a filament, a flower, a inflorescence, a leaf, amale gametophyte, a meristem, an ovary, an ovule, a petal, a pistil, apollen grain, a protoplast, a root, a seed, a sepal, a stamen, a stem, astigma, a style, a terminal bud, a cell of said plant in culture, atissue of said plant in culture, an organ, a cutting, an explant, and acallus.

GENETICALLY ENGINEERED GUAYULE PLANTS

In some alternatives of the disclosure, the guayule plants disclosedherein can include a transgene. The advent of new molecular biologicaltechniques has allowed the isolation and characterization of geneticelements with specific functions, such as sequences encoding specificprotein products or sequences having promoter activity. Scientists inthe field of plant biology developed a strong interest in engineeringthe genome of plants to contain and express foreign genetic elements, oradditional, or modified versions of native or endogenous geneticelements in order to alter the traits of a plant such as, a guayuleplant disclosed herein, in a specific manner. Any heterologous DNAsequences, whether from a different species or from the same specieswhich are inserted into the genome using genetic transformation, arereferred to herein collectively as “transgenes”.

Plant transformation involves the construction of an expression vectorwhich will function in plant cells. Such a vector typically comprisesDNA comprising a gene under control of or operatively linked to aregulatory element (for example, a promoter). The expression vector maycontain one or more such operably linked gene/regulatory elementcombinations. The vector(s) may be in the form of a plasmid, and can beused alone or in combination with other plasmids, to provide transformedguayule plants, using transformation methods as described below toincorporate transgenes into the genetic material of the guayuleplant(s).

Accordingly, in some alternatives of the disclosure, the guayule plantsdisclosed herein can include a transgene can be inserted in a nucleicacid construct which is maintained and replicated in the guayule plantas an episomal unit. In some alternatives, the transgene is stablyintegrated into the genome of the guayule plant. Stable integration canbe completed using classical random genomic recombination techniques,homologous recombination, or with more precise genome editing techniquessuch as using guide RNA directed CRISPR/Cas9 or TALEN genome editing. Insome alternatives, the transgene is present in the guayule plantdisclosed herein as a mini-circle expression vector for a stable ortransient expression.

A transgene can be introduced into a guayule plant of the presentdisclosure by a variety of techniques. These techniques, able totransform a wide variety of higher plant species including guayuleplants, are known and described extensively in the technical andscientific literature. For examples, a number of methods for planttransformation, which have been previously developed for the genetictransformation of various plant species, can be deployed for thetransformation of guayule. See, for example, Mild et al., “Proceduresfor Introducing Foreign DNA into Plants” in Methods in Plant MolecularBiology and Biotechnology, Glick B. R. and Thompson, J. E. Eds. (CRCPress, Inc., Boca Raton, 1993) pages 67-88. In addition, expressionvectors and in vitro culture methods for plant cell or tissuetransformation and regeneration of plants are readily available. See,for example, Gruber et al., “Vectors for Plant Transformation” inMethods In Plant Molecular Biology and Biotechnology, Glick B. R. andThompson, J. B. Eds. (CRC Press, Inc., Boca Raton, 1993) pp. 89-1 19.Suitable genetic transformation methods include electroporation (U.S.Pat. No. 5,384,253), micro-projectile bombardment (Sanford, J. C.Physiol Plant 7:206, 1990; Sanford I., Part. Sci. Technol. 5:27, 1987;Sanford J. C. Trends Biotech. 6:299, 1988; Klein et al., BioTechnology6:559-563, 1988; Klein, et al., Biotechnology 10:268, 1992; U.S. Pat.Nos. 5,538,880; 5,550,318; 5,736,369; and PCT Patent Pub. No. WO95/06128), Agrobacterium-mediated transformation (Moloney, et al., PlantCell Reports 8:238, 1989; Horsch et al., Science 227: 1229, 1985; Kado,Crit. Rev. Plant Sci. 10: 1, 1991; U.S. Pat. Nos. 5,563,055; 5,591,616;and EP Pat. Pub EP672752), direct DNA uptake transformation ofprotoplasts (Omirulleh et al., Plant Mol. Biol. 21(3):415-428, 1993),and silicon carbide fiber-mediated transformation (U.S. Pat. Nos.5,302,532 and 5,464,765).

More specifically, methods for the genetic transformation of guayule areknown to those of skill in the art. See, e.g., Dong et al., Plant CellRep., 25(1):26-34, 2006; U.S. Pat. No. 8,013,213; Veatch et al., Ind.Crop Prod., 22:65-74. 2005. In particular, guayule has been successfullytransformed to express several genes involved in the synthesis ofterpenoid precursors; mono-, sesqui- and di-terpenoid molecules; andisoprenoid rubber polymers using Agrobacterium-mediated transformation(Veatch et al., Industrial Crops and Products, 22:65-74, 2005). Further,methods have been developed for the optimal extraction of resin andterpenoid moieties from harvested guayule tissues (Pearson et al.,Industrial Crops and Products, 31:469-475, 2010; Salvucci et al.,Industrial Crops and Products, 30:9-16, 2009). Moreover, transgenicguayule lines have been successfully brought to field trials, where theyhave been demonstrated to accumulate increased accumulations ofterpenoid-rich resins (Veatch et al., 2005).

Additional technical details related to materials, systems and methodsuseful for genetic transformation of guayule, including selectablemarkers, suitable promoters, and expression vectors, have beenpreviously documented in, e.g., Li et al., Plant Cell Tissue Organ Cult.92: 173-181, 2008; Khemkladngoen et al., Plant Biotechnol. Rep.5:235-243, 2011; Kumar et al., Ind. Crops Prod. 32:41-47, 2010; andTsuchimoto et al., Plant Biotechnol. 29: 137-143, 2012; US Pat. Pub.Nos. US20060217512 and US20060218660, each of which is incorporatedherein by reference in its entirety.

Transformed seeds, plants, plant cells, and plant parts obtained by suchtransformation methods are intended to be within the scope of thisdisclosure. Following transformation of guayule target tissues,expression of a suitable selectable marker gene allows for preferentialselection of transformed cells, tissues and/or plants, usingregeneration and selection methods known in the art. In addition, seedsobtained from the transformed plants can be used for testing stabilityand inheritance. Generally, two or more generations are cultivated toensure that the phenotypic feature is stably maintained and transmitted.A person of ordinary skill in the art will recognizes that after atransgene is stably incorporated in transgenic plants and confirmed tobe operable, it can be introduced into other plants by sexual crossing.

Accordingly, in some alternatives of the present disclosure, a guayulehybrid plant disclosed herein can contain at least one transgene. Insome alternatives, a guayule hybrid plant disclosed herein can containat least 2, 3, 4, 5, 6, 7, 8, 9, 10 transgenes and/or no more than 15,14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 transgenes.

In addition or alternatively, in some alternatives of the disclosure,various genetic elements can be introduced into the plant genome usingtransformation techniques. These elements include, but are not limitedto genes, coding sequences, inducible, constitutive, and tissue specificpromoters, enhancing sequences, and signal and targeting sequences. Forexample, see the traits, genes and transformation methods listed in Panet al., Plant Cell, Tissue and Organ Culture, 46:2, 143-150, 1996; Donget al., Plant Cell Rep., 25: 1, 26-34, 2006; Veatch et al., Ind. CropProd., 22:65-74. 2005, and U.S. Pat. No. 8,013,213.

It will be understood by those of skill in the art that a transgene neednot be directly transformed into a plant, as techniques for theproduction of stably transformed guayule plants that pass single loci toprogeny by Mendelian inheritance is well known in the art. Such loci maytherefore be passed from parent plant to progeny plants by standardplant breeding techniques that are well known in the art. Thus, theforegoing methods for transformation would typically be used forproducing a transgenic variety. The transgenic variety could then becrossed with another (non-transformed or transformed) variety, in orderto produce a new transgenic variety. Alternatively, a genetic traitwhich has been engineered into a particular guayule variety using theforegoing transformation techniques could be moved into another varietyusing traditional backcrossing techniques that are well-known in theplant breeding ans. For example, a backcrossing approach could be usedto move an engineered trait from a public, non-elite variety into anelite variety, or from a variety containing a foreign gene in its genomeinto a variety or varieties which do not contain that gene.

Some alternatives of the present disclosure further provide a processfor producing a guayule plant that further comprises a desired trait.The process comprises transforming a guayule plant provided herein witha transgene that confers a desired trait. In some related alternatives,the disclosure provides a transformed guayule plant produced by thisprocess, and seeds produced by such transformed plants. In yet someother alternatives, the desired trait may be one or more of: abioticstress tolerance, biotic stress tolerance, disease resistance, highlatex yield, high overall rubber yield, high productivity, high resinyield, improved nitrogen use efficiency, improved water use efficiency,and plant vigor, or any combination of the aforementioned traits. Thespecific genes useful for this process may be any gene known in the artfor its ability to confer such traits. Non-limiting examples of suchtrait genes include genes encoding various allylic diphosphate synthasesin the rubber biosynthesis pathway, including geranylgeranylpyrophosphate synthase (GGPP); hexa-heptaprenyl pyrophosphate synthase,and farnesyl pyrophosphate synthase (FPP) (U.S. Pat. No. 8,013,213);Veatch et al., Ind. Crop Prod., 22:65-64. 2005; Dong et al., Plant CellRep, 25: 1, 26-34, 2006.

In a related aspect, some alternatives disclosed herein relate to seeds,plants, plant cells and plant parts disclosed herein further comprisinga transgene. In some alternatives, the disclosure further providesseeds, reproductive tissues, vegetative tissues, plant parts, biomass,or progeny of a plant according to the present disclosure.

PRODUCTS DERIVED FROM GUAYULE PLANTS

Further provided, in one aspect of the present disclosure, is a methodfor producing a plant-derived product. The method according to thisaspect of the disclosure includes obtaining a hybrid plant grown from ahybrid seed produced by any of the foregoing methods, or a part thereof,and producing said plant-derived product therefrom.

Accordingly, additionally provided, in another aspect of the presentdisclosure, is a plant product produced by a process of producing aplant-derived product disclosed herein. In some alternatives of thisaspect, the plant derived-product is selected from the group consistingof latex, resin, fatty acid triglycerides, terpenes, sesquiterpenes, orwaxes. Methods and systems useful for the production of resins derivedfrom plant species bearing rubber and rubber-like hydrocarbons have beenpreviously reported. In addition, methods and systems useful forpreparation and utilization of multi-component copolymers of guayuleresin with improved physical and chemical properties are also welldocumented. Information in this regard can be found in, for example,Ray, D. T. 1993. Guayule: A source of natural rubber, p. 338-343. In: J.Janick and J. E. Simon (eds.), New Crops. Wiley, New York; Estilai etal., Developing guayule as a domestic rubber crop, CaliforniaAgriculture, Sep.-Oct. 29-30, 1988; Ray et al., Industrial CropsProducts, 22: 15-25, 2005; Veatch et al., Ind. Crop Prod., 22.65-64.2005; Dong et al., Plant Cell Rep., 25: 1, 26-34, 2006, U.S. Pat. Pub.Nos. US20060218660, US20090163689, US20060217512, US20090099309, PCTPat. Pub. Nos. WO2007081376, WO2007136364, and WO2008147439, U.S. Pat.Nos. 5,717,050; 7,259,231; 5,580,942, 7,790,036; and 8,013,213; each ofwhich is incorporated herein by reference in its entirety.

In some preferred alternatives of this aspect, the plant derived-productis further defined as a latex product. In some particularly preferredalternatives, the latex product is selected from the group consisting ofmedical gloves, surgical gloves, elastic bands, elastic traps, condom,automobile tires, truck fires, airplane tires, and wet suits.

In some alternatives, products such as latex, resin, fatty acidtriglycerides, terpenes, sesquiterpenes, or waxes can be recovered fromthe hybrid plants of the present disclosure by recovery means known tothose skilled in the art.

All publications and patent applications mentioned in this specificationare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically canindividually indicated to be incorporated by reference.

Throughout this disclosure, various information sources are referred toand are, where specifically noted, incorporated by reference. While thecontents and teachings of each and every one of the information sourcescan be relied on and used by one of skill in the art to make and usealternatives of the disclosure, any discussion and comment in a specificinformation source should in no way be considered as an admission thatsuch comment was widely accepted as the general opinion in the field.

The discussion of the general methods given herein is intended forillustrative purposes only. Other alternative methods and aspects willbe apparent to those of skill in the art upon review of this disclosure,and are to be included within the spirit and purview of thisapplication.

Headings within the application are solely for the convenience of thereader, and do not limit in any way the scope of the disclosure or itsalternatives. Additional alternatives include the following embodiments:

1. A method for promoting the production of viable hybrid guayule seedsfrom an interploidy cross, said method comprising:

selecting a female guayule plant and a pollinator plant, wherein said afemale guayule plant is apomictic and said pollinator plant is capableof producing fertile pollen;

pollinating said female guayule plant with pollen from said pollinatorplant to produce seeds on said apomictic female guayule plant, whereinat least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40% 45%, 55%, 60%, 70%, 80%,90%, or 95% of said hybrid seeds are viable or wherein the percentage ofviable hybrid seeds produced are within a range defined by any two ofthe aforementioned percentages.

2. The method of alternative 1, wherein said pollinator plant is atetraploid guayule plant.

3. The method of alternative 1 or 2, wherein said female guayule plantis selected from a triploid guayule plant, a pentaploid guayule plant,or a heptaploid guayule plant.

4. The method of any one of alternatives 1-3, wherein said femaleguayule plant and said pollinator plant belong to the same Partheniumspecies.

5. The method of alternative 4, wherein said Parthenium species isParthenium argentatum.

6. The method of any one of alternatives 1-3, wherein said femaleguayule plant and said pollinator plant belong to two differentParthenium species.

7. The method of any one of alternatives 1-6, wherein said femaleguayule plant is a Parthenium argentatum plant.

8. The method of any one of alternatives 1-7, wherein the pollinatorplant is of a species selected from the group consisting of P. alpinum,P. argentatum, P. cineraceum, P. confertum, P. fruticosum, P.hysterophorus, P. incanum, P. integrifolium, P. ligulatum, P.rollinsianum, P. schottii, and P. tomentosum.

9. The method of any one of alternatives 1-8, wherein said pollinatorplant is a Parthenium argentatum plant or a Parthenium incanum plant.

10. The method of any one of alternatives 1-9, wherein at least one ofsaid female guayule plant or said pollinator plant is pre-selected forenhanced plant productivity.

11. The method of any one of alternatives 1-10, wherein at least one ofsaid female guayule plant or said pollinator plant is clonallypropagated prior to pollination step.

12. The method of any one of alternatives 1-11, wherein said pollinatorplant is of a Parthenium species that is different than Partheniumargentatum such that any F1 hybrid off-types from said interploidy crosscan be conveniently identified.

13. The method of any one of alternatives 1-12, wherein said percentageof viable hybrid seeds is scored at 35 days after germination.

14. A method for producing hybrid guayule seeds from an interploidycross between female guayule plants from at least one female guayuleline and pollinator plants from at least one pollinator plant line, saidhybrid seeds having a percentage of viability above a predeterminedpercentage of viability, said method comprising:

selecting said at least one female guayule line and said at least onepollinator plant line;

determining a production ratio of female plants to pollinator plants toproduce hybrid seeds having a percentage of viability above saidpredetermined percentage of viability;

planting said at least one female guayule line and said at least onepollinator plant line in pollinating proximity in said production ratioin a production field; and

allowing pollination of at least one of said female guayule plants withpollen from at least one of said pollinator plants, whereby said hybridguayule seeds are produced.

15. The method of alternative 14, further comprising harvesting saidhybrid guayule seeds.

16. The method of alternative 14 or 15, wherein said female guayuleplants comprise plants from a single female line.

17. The method of alternative 14 or 15, wherein said female guayuleplants comprise plants from a plurality of female lines.

18. The method of any one of alternatives 14-17, wherein at least one ofsaid guayule female lines is a cloned apomictic guayule line.

19. The method of any one of alternatives 14-18, wherein said pollinatorplants comprise plants from a single pollinator line.

20. The method of any one of alternatives 14-18, wherein said pollinatorplants comprise plants from a plurality of pollinator lines.

21. The method of alternative 14, wherein the predetermined percentageof viability is 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%. 45%, 55%, 60%,70%, 80%, 90, 95%, or 100% or wherein the predetermined percentage ofviability is within a range defined by any two of the aforementionedpercentages.

22. The method of any one of alternatives 14-21, wherein said percentageof viable hybrid seeds is scored at 35 days after germination.

23. The method of any one of alternatives 14-22, wherein said productionratio of female plants to pollinator plants ranges from 10:1 to 1:5.

24. The method of alternative 23, wherein said production ratio offemale plants to pollinator plants is 5:1, 4:1, 3:1, 2:1, or 1:1 orwithin a range defined by any two of the aforementioned ratios.

25. The method of any one of alternatives 14-25, wherein said selectingat least one pollinator plant line comprises selecting a pollinator linehaving high self-incompatibility or high production of viable pollen.

26. The method of any one of alternatives 14-26, wherein said plantingcomprises planting said female guayule plants and said pollinator plantsin multiple rows, wherein each row of pollinator plants is interspersedwith a plurality of rows of female guayule plants.

27. The method of alternative 26, wherein said plurality of rows offemale guayule plants is 2 to 5 rows.

28. A hybrid guayule seed produced by a method of any one ofalternatives 1-27.

29. A viable hybrid guayule seed produced by:

selecting a female guayule plant and a pollinator plant, wherein said afemale guayule plant is apomictic and said pollinator plant is capableof producing fertile pollen;

pollinating said female guayule plant with pollen from said pollinatorplant to produce seeds on said apomictic female guayule plant, whereinat least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%. 45%, 55%, 60%, 70%, 80%,90%, or 95% of said hybrid seeds are viable or wherein the percentage ofviable hybrid seeds produced are within a range defined by any two ofthe aforementioned percentages.

30. Viable hybrid seeds produced from an interploidy cross betweenfemale guayule plants from at least one female guayule line andpollinator plants from at least one pollinator plant line, said hybridseeds having a percentage of viability above a predetermined percentageof viability, wherein said hybrid seeds produced by:

selecting said at least one female guayule line and said at least onepollinator plant line;

determining a production ratio of female plants to pollinator plants toproduce hybrid seeds having a percentage of viability above saidpredetermined percentage of viability;

planting said at least one female guayule line and said at least onepollinator plant line in pollinating proximity in said production ratioin a production field; and

allowing pollination of at least one of said female guayule plants withpollen from at least one of said pollinator plants, whereby said hybridguayule seeds are produced.

31. A plant grown from the hybrid guayule seed of any one ofalternatives 28-30.

32. The plant of alternative 31, further comprising a transgene.

33. The plant of alternative 32, wherein said transgene confers a traitselected from the group consisting of: abiotic stress tolerance, bioticstress tolerance, disease resistance, high latex yield, high overallrubber yield, high productivity, high resin yield, improved nitrogen useefficiency, improved water use efficiency, plant vigor, and combinationsof at least two of the aforementioned traits.

34. A seed, a reproductive tissue, a vegetative tissue, a plant part, abiomass, or a progeny of a plant according to any one of alternatives31-33.

35. A plant part of alternative 34, wherein said plant pan is selectedfrom the group consisting of an anther, an axillary bud, a cell, anembryo, a female gametophyte, a filament, a flower, a inflorescence, aleaf, a male gametophyte, a meristem, an ovary, an ovule, a petal, apistil, a pollen grain, a protoplast, a root, a seed, a sepal, a stamen,a stem, a stigma, a style, a terminal bud, a cell of said plant inculture, a tissue of said plant in culture, an organ, a cutting, anexplant, and a callus.

36. A method for producing a product, comprising obtaining a plant orparts thereof set forth in any one of alternatives 31-35.

37. A plant derived-product produced by the method of alternative 36.

38. The plant derived product of alternative 37, which is selected fromthe group consisting of latex, resin, fatty acid triglycerides,terpenes, sesquiterpenes, and waxes.

39. A latex product derived from the latex of alternative 38, whereinsaid. latex product is selected from the group consisting of medicalgloves, surgical gloves, elastic bands, elastic traps, condom,automobile tires, truck tires, airplane tires, and wet suits.

EXAMPLES

Additional alternatives are disclosed in further detail in the followingexamples, which are not in any way intended to limit the scope of theclaims.

Example 1 Production of Triploid Guayule Offspring via 2××4× InterploidyPollination

This Example illustrates methods for producing triploid offspring viapollination of a diploid female guayule plant with pollen from atetraploid pollen donor. In a first set of experiments, fluorescent flowcytometry was used to analyze the nucleic acid content and ploidy levelof hybrid seeds derived from 2××4× interploidy crosses. Flow cytometryhas been used previously for cytological applications such asdetermination of ploidy levels, detection of aneuploidy, screening ofreproductive mode, analysis of cell cycle, and estimation of absolutenuclear DNA amount (nuclear genome size). Compared to chromosomecounting, flow cytometry is a rapid and simple method for thedetermination of ploidy levels in crop species. With a high level ofaccuracy, flow cytometry indirectly estimates the number of chromosomesin somatic cells by measuring the DNA content of fluorescence stainednuclei. Further information regarding applications of this technique indetermination of guayule ploidy levels can be found in, for example,Gore et al., Crop Science, 51:210-216, 2011, hereby incorporated byreference in its entirety.

Four tetraploid and four diploid guayule plants were grown understandard greenhouse conditions until flowering. Each diploid plant waspruned of flowers and placed in isolation within a pollination cagetogether with one of the tetraploid plants. Blue bottle flies (genusCalliphora) were added weekly to the cages to facilitate pollination.Achenes were harvested from each diploid plant after thirty days ofco-cultivation. The achenes were then germinated on a paper towel in a15-cm petri dish with 20 ml of diH₂O. The petri dishes were sealed withparafilm and incubated in a growth chamber with a 14 hour day length,day temperature of 27° C. and night temperature of 22° C. forgermination. The number of germinated seeds was scored at day 4, day 7,and day 14 of incubation. Seedlings were transferred to grow containers(Grodan™ blocks) a few days following germination.

After the seedlings were fully established in 4-inch pots, two 5-mm holepunches were sampled from each seedling in triplicate and placed into a96-well microtube rack. Tissue was also collected from plants ofdiploid, triploid, and tetraploid plant controls and loaded on themicrotube rack. A 3-mm carbide bead and 500 μL of Baranyi I solution.(100 mM citric acid monohydrate; 0.5% (v/v) Triton X-100) were loadedinto each well before disrupting the samples with a TissueLyser IIsolution (QIAGEN) at 27 Hz for 30 seconds. Nuclear lysates were thencentrifuged through a 30-40 um filter to remove cellular debris.Subsequently, two parts Baranyi II solution (400 mM Na₂HPO₄, 10 mMsodium citrate, 25 mM sodium sulfate) mixed with SYBR® Green I(Sigma-Aldrich) were added to one part of the nuclei extract to bringthe final sample to 2×SYBR Green I and a neutral PH (7.0-8.0). Stainednuclei samples were allowed to incubate in the dark at room temperaturefor 30 minutes before analysis. The fluorescence value relative to theDNA content of nuclei for each sample was acquired using an Attune® FlowCytometer (Life Technologies) and Attune® Autosampler (LifeTechnologies). An acquisition flow rate of 100 μl/min and an acquisitionvolume of 50 μl were used. Nuclei peaks were manually gated (FIGS. 1 and2 ) and the resulting median fluorescence values were compared againstintraspecific controls to generate ploidy callus.

Germination rates were scored after 35 days of incubation. As summarizedin TABLE 1, the scored germination rates of seeds derived from 2××4×interploidy crosses varied from 13.4% to 39 4%. The percentages ofseedling survival after 35 days of the seedlings that germinated were35.7% and 65.6%, respectively. Notably, a significant fraction ofseedlings derived from the interploidy crosses, if not all seedlings.germinated within the first 7 days.

TABLE 1 Germination results from each diploid × tetraploid (2X × 4X)caged cross. Achenes were collected from the diploid in each crossNumber of Number of Achenes Seedling Survival @ Collected GerminatedPervent Germination Day 35 Location Cross Achenes Day 4 Day 7 Day 14 Day4 Day 7 Day 14 Number Percent CAGE 2 2X × 4X  71 20 28 28 28.2 39.4 39.410 35.7 CAGE 3 2X × 4X 170 32 45 45 18.8 26.5 26.5 25 55.5 CAGE 4 2X ×4X 175 32 40 40 18.3 22.9 22.9 18 45   CAGE'S 2X × 4X 239 30 30 32 12.612.6 13.4 21 65.6

Fluorescence data acquired from each F1 seedling was plotted alongsidedata from other F1 seedlings of the same caged cross (FIG. 3 ). Eachcolumn represents a sample replicate corresponding to a particular F1seedling. Values were ordered from lowest to highest fluorescenceintensity and showed only small variation. Fluorescence values for theintraspecific controls are plotted as horizontal dotted lines. As shownin TABLE 2, three of the four 2××4× interploidy crosses produced onlytriploid F1 seedlings. It was also observed that one of the crosses(Cage 3) produced 4 diploids, 18 triploids, and 1 tetraploid, asindicated by the fluorescence data plotted for Cage 3, which clearlyshowed separation between the seedling samples of different ploidylevels.

TABLE 2 Ploidy levels of the F1 seedlings derived from each of the fourinterploidy crosses. F1 Seedlings Diploid, Triploid, Tetraploid, %Location 2X 3X 4X Total Hybrid CAGE 2 0 7 0 7 100% CAGE 3 4 18 1 23  78%CAGE 4 0 16 0 16 100% CAGE 5 0 20 0 20 100%

Conclusions

The ploidy levels of the F1 seedlings from each caged cross indicatedthat pollination of a diploid female plant with pollen from a tetraploidpollen donor produced predominantly triploid (hybrid) offspring. Whilethree out of the four caged crosses produced only triploid offspring,the cross performed in Cage 3 produced unexpected offspring of varyingploidy. While the inventors do not wish to be bound by any theory, it isbelieved that the variation in the offspring may be due to the geneticsof either of the parents involved in the cross.

Example 2 Production of Viable Seeds from Triploid Apomictic P.Argentatum Plant in the Presence of a Fertile Pollen Source

This Example illustrates methods for promoting the production of viableseeds from an apomictic guayule plant in accordance with at least somealternatives of the present disclosure.

Two genetically identical triploid P. argentatum plants and twotetraploid P. argentatum plants were grown under standard greenhouseconditions until flowering. One of the triploid plants was grown inisolation and allowed to self-pollinate to produce selfed-seeds. Theother triploid plant was spatially isolated from the first triploidplant and was grown alongside with the two tetraploid plants. Acheneswere harvested from each of the triploid plants after thirty days ofisolated growth. The achenes were then germinated on a paper towel in a15 cm petri dish with 20 ml of diH₂O. The petri dishes were sealed withparafilm and incubated in a growth chamber with a 14 hour day length,day temperature of 27° C. and night temperature of 22° C. The number ofgerminated seeds was scored at day 4, day 7, and day 14 of incubation.Seedling survival scores were revisited at day 35 of incubation. Theresults of this germination study are summarized in TABLE 3.

TABLE 3 Germination counts, percentages, and survival rates of the twogeographically isolated crosses Achenes Number of Number of AchenesPercent Seedling Survival @ Collected Achenes Germinated Germination Day35 Cross From Collected Day 4 Day 7 Day 14 Day 4 Day 7 Day 14 NumberPercent 3X × 4X 3X 54 7 14 16 12.9 25.9 29.6 12 75 3X 3X 66 0  3  6 0 4.5   9.1  0  0 selfed

Seedlings resulting from the pollination of the triploid plant withpollen from a tetraploid showed a 75% survival rate at 35 days. Incontrast, the seedlings resulting from the selfing of a geneticallyidentical triploid plant showed no survival, indicating that fertilepollen is necessary to produce viable offspring from triploid plants.Although six seedlings from the selfing of the second triploid plant didinitially germinate, none survived beyond just a few days and each wasobserved to display abnormal growth patterns. Without being bound to anyparticular theory, it may well be that the development of embryo and/orendosperm did not complete in these seeds. This germination studyindicates that the presence of fertile pollen is needed to produceviable seed from triploid apomictic guayule.

Example 3 Interspecific Pollination Promotes Production of Viable Seedsfrom Apomictic Pentaploid Parthenium Argentatum

This Example illustrates methods in accordance with at least somealternatives of the present disclosure, in which fertile pollen ofmariola (P. incanum) can be used as a pollen donor for the production ofviable seeds from an apomictic P. argentatum plant.

A mariola plant and pentaploid guayule plant were grown under standardgreenhouse conditions until flowering. The plants was pruned of flowersand placed together in a pollination cage. Blue bottle flies (genusCalliphora) were added weekly to the cages to facilitate pollination.Achenes were harvested from the guayule plant after thirty days ofco-cultivation. The achenes were then germinated on a paper towel in a15 cm petri dish with 20 ml of diH₂O. The petri dish was sealed withparafilm and incubated in a growth chamber with a 14 hour day length,day temperature of 27° C. and night temperature of 22° C. forgermination. The number of germinated seeds was scored at day 4, day 7,and day 14. Seedling survival was revisited at day 35 of incubation. Theresults of this germination study are summarized in TABLE 4. Remarkably,7.5% of the seeds germinated and of these, 58.3% of the seedlingsderived from the interploidy crosses germinated and survived after 35days of incubation.

TABLE 4 Germination counts, percentages, and survival rates of F1hybrids of the two caged crosses Achenes Number of Number of AchenesPercent Seedling Survival Collected Achenes Germinated Germination @ Day35 Location Cross From Collected Day 4 Day 7 Day 14 Day 4 Day 7 Day 14Number Percent CAGE 7 5X × Mariola 5X 159 0 9 12 0 5.7 7.5 7 58.3

After the F1 seedlings were fully established in 4-inch pots, two 5-mmhole punches were sampled from each seedling in triplicate and placedinto a 96-well microtube rack. Tissue was also collected from plants ofdiploid, triploid, and tetraploid plant controls and loaded on themicrotube rack. Ploidy levels of the hybrid seedlings were determined byusing the same procedure described in Example 1 above. Nuclei peaks weremanually gated (FIGS. 4 and 5 ) and resulting median fluorescence valueswere compared against intraspecific controls to generate ploidy callus.

The F1 seedling fluorescence data (FIG. 7 ), when compared againstintraspecific controls (FIG. 6 ), indicates that all six F1 seedlingswere pentaploids.

Conclusions

As previously shown, and described in Example 2 above, hybrid guayulerequires a source of fertile pollen to produce viable seeds. In Example3, seeds were germinated from a cross between guayule and mariola, andthe experiments showed that seedlings derived from these hybrid seedswere viable. The results indicated that mariola could be used as apollen donor to produce viable guayule seeds from apomictic pentaploidplants.

Subsequent cytometric analysis revealed that these hybrid seedlings wereall pentaploid, indicating that they were apomictic progeny of themother pentaploid plant. Additionally, these seedlings displayed nophenotypic features characteristics of interspecific hybrids. Theseplants are expected to have been produced apomictically, which can beverified by Genotyping-by-Sequencing.

Comparative experiments with diploid and tetraploid mariola used aspollen donors are contemplated so as to determine which ploidy will bemost effective as a pollinator. Additionally, it is contemplated thatone can produce higher seed counts from these interploidy and/orinterspecific crosses so as to increase the probability of producinginterspecific off-types resulting from the fertilization of the guayuleembryo by mariola pollen, as this may help to validate the approach ofculling by interspecific phenotype during seed multiplication. It isfurther contemplated that one can produce higher seed counts from theseinterploidy and/or interspecific crosses so as to increase the rate ofseed increase.

Although initially mariola was used as a pollinator because it is themost closely related species to guayule, the methods described hereincan be used with any of the 14 other species in the Parthenium genus aspollinators.

While particular alternatives of the present disclosure have beendisclosed, it is to be understood that various modifications andcombinations are possible and are contemplated within the true spiritand scope of the appended claims. There is no intention, therefore, oflimitations to the exact abstract and disclosure herein presented.

What is claimed is:
 1. A method for producing hybrid guayule seeds froman interploidy cross between female guayule plants from at least onefemale guayule line and pollinator plants from at least one pollinatorplant line, said hybrid seeds having a percentage of viability above apredetermined percentage of viability, said method comprising: selectingsaid at least one female guayule line and said at least one pollinatorplant line; determining a production ratio of female plants topollinator plants to produce hybrid seeds having a percentage ofviability above said predetermined percentage of viability; plantingsaid at least one female guayule line and said at least one pollinatorplant line in pollinating proximity in said production ratio in aproduction field; and allowing pollination of at least one of saidfemale guayule plants will pollen from at least one of said pollinatorplants, whereby said hybrid guayule seeds are produced.
 2. A plant grownfrom a hybrid guayule seed produced by the method of claim
 1. 3. Theplant of claim 2, further comprising a transgene.
 4. The plant of claim3, wherein said transgene confers a trail selected from the groupconsisting of: abiotic stress tolerance, biotic stress tolerance,disease resistance, high latex yield, high overall rubber yield, highproductivity, high resin yield, improved nitrogen use efficiency,improved water use efficiency, plant vigor, and combinations of at leasttwo of the aforementioned traits.